Generation of cCdc14A-KO and cCdc14B-KO cells
cCdc14A and cCdc14B cDNAs were isolated by RT-PCR using total RNA extracted from DT40 WT cells as template. For cCdc14A, two separate targeting vectors containing a 2.3-kb left and 3-kb right arm of homology were synthesized and cloned into Bluescript (Agilent Technologies) flanking either puromycin or blasticidin selection cassettes (Sonoda et al., 1998
). After one round of targeting with the puromycin-targeting vector, drug-resistant clones were genotyped by PCR to identify cCdc14A+/−
cells. After excision of the puromycin cassette by induction of Cre recombinase, one hemizygous clone was electroporated with a rescue vector containing the HA-tagged cCdc14A cDNA under control of the chicken β-actin promoter and a puromycin-resistant cassette (plasmid derived from pBluescript; provided by J.M. Buerstedde, Institute for Molecular Radiobiology, Munich, Germany) and selected in 0.5 µg/ml puromycin (InvivoGen). Clones expressing cCdc14A at close to endogenous levels were electroporated with the targeting vector as described previously (Saribasak and Arakawa, 2006
) and selected in 30 µg/ml blasticidin to obtain a nullizygous clone. To generate the cCdc14A rescue cell line, cCdc14A-KO cells were transfected with the cCdc14A vector and selected in 0.5 µg/ml puromycin. Drug-resistant clones were screened for cCdc14A expression by IB, and a cell line with nearly endogenous levels of cCdc14A was identified and designated cCdc14A-Res.
For cCdc14B, a targeting vector containing a 3.3-kb left and 4.8-kb right arm of homology was synthesized by long-range PCR and cloned into Bluescript flanking a blasticidin selection cassette (Sonoda et al., 1998
). To generate cCdc14B-deficient DT40 clones, DT40-Cre-ER cells expressing cCdc14B-HA were transfected with the targeting construct, using initially PCR and subsequently Southern blotting to genotype drug-resistant clones after selection in 30 µg/ml blasticidin (InvivoGen). To generate the cCdc14B rescue cell line, cCdc14B-KO cells were transfected with a rescue vector containing the HA-tagged cCdc14B cDNA under control of the chicken β-actin promoter and a puromycin-resistant cassette and selected in 0.5 µg/ml puromycin. Drug-resistant clones were screened for cCdc14B-HA expression by IB, and the positive clones designated cCdc14B-Res.
Generation of hCdc14A-KO cells
To generate a conditional KO of the hCdc14A locus, 5′ and 3′ homology arms were amplified from a human BAC clone (RP11-976L7) and cloned into a vector containing a central FRT-neo-FRT-loxP cassette. A secondary loxP site was introduced downstream of exon 2 via QuikChange mutagenesis. The entire Cdc14A insert was subcloned into pAAV. Transfection of HEK293 cells, isolation of AAV particles, and infection of hTERT-RPE cells were performed as described previously (Berdougo et al., 2009
). G418-resistant colonies were screened by PCR. The neo cassette was excised from Cdc14Aflox-neo/+
cells by transfection with pCAAGS-FLPe followed by puromycin selection and limiting dilution. Individual colonies were tested for neo excision by genomic PCR and reacquisition of G418 sensitivity. Targeting of the second allele was achieved with a Cdc14A vector lacking exon 2 (pAAV-Cdc14AΔ). Cdc14Aflox/Δneo
cells were converted to Cdc14AΔ/Δneo
cells by infection with a recombinant adenovirus-expressing Cre recombinase. Targeted clones were confirmed by Southern blotting. The transcript from the exon 2–deleted hCdc14A
gene contains a frameshift and does not code for a functional hCdc14A protein.
Cell culture and treatments
DT40 B-lymphoma cells DT40 B-lymphoma cells were grown in DME (Invitrogen) containing 10% FBS, 1% chicken serum, 1% glutamine, 1% sodium pyruvate, 10−5 M β-mercaptoethanol, penicillin, and streptomycin at 40°C. HCT116 cells were grown in McCoy’s 5A medium (Invitrogen) supplemented with 10% FBS (Invitrogen) at 37°C. hTERT-RPE1 cell lines were grown in DME/F-12 medium supplemented with 10% FBS, 1% glutamine, and 0.348% sodium bicarbonate at 37°C.
Cells were irradiated with 10 Gy IR using a caesium source (Gamma Cell 1000; Atomi Energy of Canada Ltd) and treated with 0.5 µg/ml Noco (Sigma-Aldrich), 5 µM aphidicolin (Sigma-Aldrich), 2 mM thymidine (Sigma-Aldrich), 5 mM caffeine (Sigma-Aldrich), 1.5 µM DXR (Applichem), and 0.1 mM 4-hydroxytamoxifen (Sigma-Aldrich) as appropriate.
Cells were fixed in 70% ethanol in PBS overnight. For DNA content analysis, cells were pelleted and resuspended in PBS containing 1 mg/ml RNase (Sigma-Aldrich) and 10 mg/ml propidium iodide (PI) incubated at room temperature for 30 min then analyzed using a flow cytometer (FACScan; BD).
For MI determinations, fixed cells were incubated with polyclonal anti–phospho histone H3 antibodies followed by FITC-conjugated secondary antibody (Invitrogen). Cells were counterstained with propidium iodide and analyzed for FITC fluorescence and DNA content by flow cytometry.
For determination of γ-H2A.X foci, fixed cells were incubated with monoclonal anti–γ-H2A.X antibody followed by FITC-conjugated secondary antibody and counterstained with propidium iodide.
Cell extracts were prepared in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 50 mM Tris-Cl, pH 8.0, 1 mM PMSF, complete protease inhibitor cocktail [Roche], and PhosStop phosphatase inhibitor cocktail [Roche]), resolved by SDS-PAGE, and blotted onto nitrocellulose membranes (GE Healthcare). Antibodies against Chk1(S345ph) (Cell Signaling Technology), Chk1 (G-4; Santa Cruz Biotechnology, Inc.), Cdk1(Y15ph) (IL-15; Santa Cruz Biotechnology, Inc.), and Cdk1 (cl 17; Santa Cruz Biotechnology, Inc.) were used for IB. A polyclonal rabbit antiserum specific for avian cCdc14A was generated against the C-terminal 257 amino acids of the protein. The antibody against Chk2 was described previously (Zachos et al., 2003
). Blots were scanned using a luminescence fluorimager (LAS4000; Fujifilm) and quantified using Multi Gauge software (Fujifilm).
IF and microscopy
Antibodies against γ-tubulin (GTU-88; Sigma-Aldrich), cCdc14A, GFP (purified in house), fibrillarin (4G9-E4; Cytoskeleton, Inc.), B23 (C-19; Santa Cruz Biotechnology, Inc.), γ-H2A.X(S139) (Millipore), and pH3(S10) (Millipore) were used for IF. In brief, cells were either grown on coverslips or allowed to attach to polylysine slides (VWR International), fixed with 4% paraformaldehyde for 10 min at 37°C, permeabilized with PBS-T (PBS + 0.1% Triton X-100), and blocked with 10% FBS in PBS-T for 30 min at 37°C before application of primary antibody. Alternatively, cells were fixed in 100% methanol at −20°C for 5 min. Alexa Fluor 488– and 594–conjugated secondary antibodies (Invitrogen) were used. For the detection of γ-H2A.X foci, cells were fixed with 3.7% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 10% fetal calf serum and 0.5% bovine serum albumin in PBS for 30 min. Anti-pH3 and –γ-H2A.X were diluted 1:100 in blocking buffer. Cells were incubated with the antibodies for 60 min and washed three times for 5 min in blocking buffer. Anti–rabbit Alexa Fluor 594 and anti–mouse Alexa Fluor 488 (Invitrogen) were each used at 1:500 dilution in blocking buffer. Cells were incubated with the secondary antibodies for 60 min, washed twice for 5 min with blocking buffer, and once for 5 min with PBS before being mounted in ProLong gold (Invitrogen).
Images were taken on a microscope (DeltaVision RT; Applied Precision) equipped with GFP and TRITC filters (Chroma Technology Corp.), a Plan Apo 100× NA 1.4 oil immersion objective (IX70; Olympus), softWoRx software (Applied Precision), and a camera (CoolSNAP HQ; Photometrics). Image stacks were deconvolved and projected using softWoRx.
Single-cell gel electrophoresis (alkaline comet) assay
Single-cell comet assays were performed as per the manufacturer’s instructions (Trevigen). Nuclei were visualized using epifluorescent illumination on a microscope (Carl Zeiss, Inc.), and images were analyzed using ImageJ software (National Institutes of Health).
Cell viability assay
Treated or untreated cells were seeded in octuplicate microtiter wells at 5 × 103 cells/well for hTERT-RPE1 and HCT116 or at 105 cells/well for DT40, incubated overnight, and irradiated or grown in medium for 48 h or 24 h, respectively. Viability was measured by method of transcriptional and translational (MTT) assay. Results were expressed as the OD550 relative to that of untreated cells.
Online supplemental material
Fig. S1 shows localization of cCdc14A and cCdc14B, cCdc14A-KO strategy, and confirmation of KO. Fig. S2 shows cCdc14B-KO strategy, confirmation of cCdc14B-KO cells, and absence of growth defects in cCdc14B-KO cells. Fig. S3 shows localization of cCdc14A and cCdc14B after IR and absence of adaptation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200910057/DC1